Cellular phone based optical detection of specific nucleic acid sequences

ABSTRACT

A technique relates to determining a presence of a known nucleic acid sequence of a known molecule in a sample. A sample surface is coated with a select segment of the known molecule. Beads are coated with a first molecule. A targeted nucleic acid sequence is attached to a second molecule that binds to the first molecule, such that the targeted nucleic acid sequence is attached to the beads via first and second molecules. The sample is placed on the sample surface. The sample includes the liquid medium, beads, and targeted nucleic acid sequence being tested. Brownian motion of beads is monitored to determine whether the Brownian motion of the beads is restricted or not restricted. When the Brownian motion of beads is restricted, the presence of the known nucleic acid sequence is in the sample, thus indicating that the targeted nucleic acid sequence is the known nucleic acid sequence.

DOMESTIC PRIORITY

This application is a continuation of U.S. patent application Ser. No.14/725,668, filed May 29, 2015, the disclosure of which is incorporatedby reference herein in its entirety.

BACKGROUND

The present invention relates to nucleic acid sequences, and morespecifically, to optical detection of specific nucleic acid sequences.

A nucleic acid sequence is a succession of letters that indicate theorder of nucleotides within a deoxyribonucleic acid (DNA) (using G, A,C, and T) or ribonucleic (RNA) (G, A, C and U) molecule. By convention,sequences are usually presented from the 5′ end to the 3′ end.

Deoxyribonucleic acid (DNA) is a molecule that encodes the geneticinstructions used in the development and functioning of all known livingorganisms and viruses. Ribonucleic acid (RNA) is a polymeric molecule.It is implicated in various biological roles in coding, decoding,regulation, and expression of genes.

SUMMARY

According to one embodiment, a method is provided to determine apresence of a known nucleic acid sequence of a known molecule in asample in which the sample contains a targeted nucleic acid sequence.The method includes coating a sample surface with a select segment ofthe known molecule, where the sample surface is configured to hold aliquid medium, and coating a plurality of beads with a first molecule.The method includes attaching the targeted nucleic acid sequence to asecond molecule that is configured to bind to the first molecule, suchthat the targeted nucleic acid sequence is attached to the plurality ofbeads via the first and second molecules, and placing the sample on thesample surface. The sample includes the liquid medium, the plurality ofbeads, and the targeted nucleic acid sequence being tested for havingthe known nucleic acid sequence of the known molecule. The targetednucleic acid sequence is unknown. Also, the method includes videomonitoring a Brownian motion of the plurality of beads with an opticaldevice, and determining whether the Brownian motion of the plurality ofbeads in the liquid medium is restricted or not restricted over time. Inresponse to determining that the Brownian motion of the plurality ofbeads is restricted, the presence of the known nucleic acid sequence isrecognized to be in the sample, thus indicating that the targetednucleic acid sequence is the known nucleic acid sequence. In response todetermining that the Brownian motion is not restricted, it is recognizedthat the known nucleic acid sequence is not present in the sample, thusindicating that the targeted nucleic acid sequence is not the knownnucleic acid sequence.

According to one embodiment, a system is provided to determine apresence of a known nucleic acid sequence of a known molecule in asample in which the sample contains a targeted nucleic acid sequence.The system includes a sample surface configured to receive the sample,and the sample surface is coated with a select segment of the knownmolecule. The sample includes a plurality of beads coated with a firstmolecule, and the targeted nucleic acid sequence attached to a secondmolecule that is configured to bind to the first molecule, such that thetargeted nucleic acid sequence is attached to the plurality of beads viathe first and second molecules. The targeted nucleic acid sequence isbeing tested for having the known nucleic acid sequence of the knownmolecule, and the targeted nucleic acid sequence is unknown. The samplealso includes a liquid medium. Also, the system includes an electroniccommunication device configured to video monitor a Brownian motion ofthe plurality of beads, and determine whether the Brownian motion of theplurality of beads in the liquid medium is restricted or not restrictedover time. The electronic communication device is configured to inresponse to determining that the Brownian motion of the plurality ofbeads is restricted, recognize the presence of the known nucleic acidsequence in the sample, thus indicating that the targeted nucleic acidsequence is the known nucleic acid sequence. Also, the electroniccommunication device is configured to in response to determining thatthe Brownian motion is not restricted, recognize that the known nucleicacid sequence is not present in the sample, thus indicating that thetargeted nucleic acid sequence is not the known nucleic acid sequence.

According to one embodiment, an electronic communication device isprovided to determine a presence of a known nucleic acid sequence of aknown molecule in a sample in which the sample contains a targetednucleic acid sequence. The electronic communication device includes aprocessing circuit and a non-transitory storage medium readable by theprocessing circuit and storing instructions that, when executed by theprocessing circuit, cause the processing circuit to perform operations.The processing circuit performs operations of video monitoring of aBrownian motion of a plurality of beads attached to the targeted nucleicacid sequence, and determining whether the Brownian motion of theplurality of beads in a liquid medium is restricted or not restrictedover time. The processing circuit performs operations of in response todetermining that the Brownian motion of the plurality of beads isrestricted, recognizing the presence of the known nucleic acid sequencein the sample, thus indicating that the targeted nucleic acid sequenceis the known nucleic acid sequence. The processing circuit performsoperations of in response to determining that the Brownian motion is notrestricted, recognizing that the known nucleic acid sequence is notpresent in the sample, thus indicating that the targeted nucleic acidsequence is not the known nucleic acid sequence.

Additional features and advantages are realized through the techniquesof the present invention. Other embodiments and aspects of the inventionare described in detail herein and are considered a part of the claimedinvention. For a better understanding of the invention with theadvantages and the features, refer to the description and to thedrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The subject matter which is regarded as the invention is particularlypointed out and distinctly claimed in the claims at the conclusion ofthe specification. The forgoing and other features, and advantages ofthe invention are apparent from the following detailed description takenin conjunction with the accompanying drawings in which:

FIG. 1 illustrates a sample surface with a liquid on the sample surfaceaccording to an embodiment;

FIG. 2 is a cross-sectional view of a system utilized for detection of aspecific DNA sequence within an unknown DNA molecule being testedaccording to an embodiment;

FIG. 3A is a top-down view illustrating that a tethered bead attachedvia the unknown DNA molecule is confined to motion in a circular shapeaccording to an embodiment;

FIG. 3B is graph illustrating the effects of DNA tethering on x and ybead displacement according to an embodiment;

FIG. 3C is graph illustrating the effects of DNA tethering mean-squareddisplacement according to an embodiment;

FIG. 4 is a system illustrating a cellular phone based bead motionimaging microscope according to an embodiment;

FIG. 5 is a system illustrating a cellular phone based bead motionimaging microscope according to another embodiment;

FIG. 6 illustrates a Wollaston-like doublet arrangement with twohalf-ball lenses according to an embodiment;

FIG. 7A is an image illustrating polystyrene beads of micron size stuckon a glass slide according to an embodiment;

FIG. 7B is an image pre-processed after converting to a gray-scale imageand band pass filtering for optimal feature enhancement according to anembodiment;

FIG. 7C is an image of bead positions detected with a MATLAB® functionaccording to an embodiment;

FIG. 7D is a histogram graph illustrating the distribution of thedetected circle radius in pixels from the image in FIG. 7C according toan embodiment;

FIGS. 7E-1 and 7E-2 are histogram graphs illustrating bead displacementin bead positions between two consecutive video frames for x-positionsand y-positions, respectively, according to an embodiment;

FIGS. 7F-1 and 7F-2 are histogram graphs illustrating bead displacementin bead positions between two consecutive video frames for x-positionsand y-positions, respectively, according to an embodiment;

FIGS. 7G-1 and 7G-2 are histogram graphs illustrating bead displacementin bead positions between two consecutive video frames for x-positionsand y-positions, respectively, according to an embodiment;

FIG. 8A-1 is an original fluorescent image illustrating micron sizebeads according to an embodiment;

FIG. 8A-2 is a processed fluorescent image of the micron size beadsidentifying bead positions between two consecutive video frames;

FIG. 8B is a graph illustrating pixel-level intensity distribution of adetected bead image according to an embodiment;

FIG. 8C-1 is an image illustrating bright spots identified as beadpositions according to an embodiment;

FIG. 8C-2 is a histogram graph illustrating the distribution of foundcircle radii in pixels for the image in FIG. 8C-1 according to anembodiment;

FIG. 8D-1 is a histogram graph of the distribution of total displacementin bead positions between two consecutive video frames for x-positionsaccording to an embodiment;

FIG. 8D-2 is a histogram graph of the distribution of total displacementin bead positions between the two consecutive video frames fory-positions according to an embodiment;

FIG. 8E-1 is a histogram graph of the distribution of totalx-displacement in bead positions between two consecutive video framesaccording to an embodiment;

FIG. 8E-2 is a histogram graph of the distribution of totaly-displacement in bead positions between two consecutive video framesaccording to an embodiment;

FIG. 9A is an image of a frame illustrating the bead motion according toan embodiment;

FIG. 9B-1 is the band-pass filtered image and identified bead positionsaccording to an embodiment;

FIG. 9B-2 is a zoomed in view of the band-pass filtered image accordingto an embodiment;

FIG. 9C-1 is a histogram graph illustrating the distribution beadpositions for x-positions as shifted between two frames which fits aGaussian distribution as expected for the random displacement of thebeads according to an embodiment;

FIG. 9C-2 is a histogram graph illustrating the distribution beadpositions for x-positions as shifted between two frames which fits aGaussian distribution as expected for the random displacement of thebeads according to an embodiment;

FIG. 9C-3 is a histogram graph illustrating the distribution beadpositions for y-positions as shifted between two frames which fits aGaussian distribution as expected for the random displacement of thebeads according to an embodiment;

FIGS. 10A and 10B together illustrate a process of optical detection todetermine the absence and/or presence of a nucleic acid sequence in asample being tested according to an embodiment;

FIGS. 11A and 11B together illustrate a method to determine apresence/absence of a known nucleic acid sequence of a known molecule ina sample according to an embodiment;

FIG. 12 illustrates an example of a computer system/phone programed toexecute the features of embodiments; and

FIG. 13 is a cross-sectional view of a system utilized for detection ofa specific DNA sequence within an unknown DNA molecule being testedaccording to an embodiment; and

FIG. 14 is a cross-sectional view of a system utilized for detection ofa specific DNA sequence within an unknown DNA molecule being testedaccording to an embodiment.

DETAILED DESCRIPTION

Embodiments provide techniques to detect the presence or absence of aspecific nucleic acid sequence, such as DNA and RNA, in atargeted/unknown molecule being tested.

There are times when one needs to know whether a sample, which may beany item or substance being tested, contains a known nucleic acidsequence. For example, Europe and now the U.S. have begun requiringlabelling of genetically modified (GM) food. One may not know whether ashipment of corn was actually from non-GM plants or GM plants. Inanother scenario, individuals with Celiac disease become sick if theyeat gluten. How would a person know if this ‘gluten-free’ product reallycontains gluten? It is possible that products can be fraudulent orhealth hazards but individuals may not know.

Embodiments provide techniques to detect the presence or absence of aspecific nucleic acid sequence, such as DNA and RNA, in a sample beingtested. Accordingly, embodiments can identify the presence of pathogens,viruses, organisms, particular DNA and RNA molecules, etc., usingoptical detection on a device, such as, e.g., a cellular phone (alsoreferred to as mobile phones, smart phones, mobile devices, etc.).

Embodiments are based on detecting the restricted Brownian motion ofmicroparticles which can be applied to detect the genetic material(DNA/RNA) of flu and other viruses, bacteria, and other organisms.Because embodiments are based on the complementarymetal-oxide-semiconductor (CMOS) sensor in a mobile phone (or otherdevice), some embodiments can be fabricated on a silicon chip withintegrated optical components to make low-cost, compact, and portablebiosensing chips. Due to their portability and compactness, thesebiosensing chips can have wide applicability in the developing countrieswhere quick and fast diagnosis can be made with these devices whileusing limited resources.

The current mobile phone user market is huge and the number of mobilephone users has reached 5 billion in 2010. The majority of these usersare in the developing countries. Moreover, it is estimated that therewill be 8 billion smart phone users with connected health and wellnessservice by 2018. This scenario offers a tremendous opportunity fordeveloping smart-phone based biosensors delivering health care relatedinformation, for example, identifying if a person has the flu or not,performing a cholesterol test, and so on. Smart phones are built with agood complementary metal-oxide-semiconductor (CMOS) sensors having highimage capturing and processing capabilities, for example iPhone® 5, 5C,and 6, and this offers a great opportunity for use in embodimentsdiscussed herein. There is a growing need for quick and easy diagnosisof flu-like symptoms with point-of-care diagnostic kits at one's home,away from the doctor's office, or at an epidemic center. This is alsohighlighted by the recent epidemic associated with the Ebola virus.There have been recent advances made by researchers in developingmobile-phone based sensing devices, for example, for flow cytometryanalysis and polymerase chain reaction (PCR) based DNA detection.However, embodiments apply techniques using the Brownian motion ofmicroparticles to detect the presence of DNA or RNA molecules specificto a known DNA sequence such as, for example, the flu type virus.

For ease of understanding, sub-headings are utilized. It is understoodthat the sub-headings are meant for explanation purposes and notlimitation.

Brownian Motion

Colloidal or polymer particles of micron size undergo random motion in aliquid (also referred to as a medium or solution), and this randommotion is characteristic of the Brownian motion. Brownian motion canalso be observed in air, for example, by watching small pollen grains ordust particles suspended in air shined by a beam of sun light. In bothcases, the origin of their random motion can be explained by the randommotion of the underlying much smaller molecules that make up either theair or liquid. As these small molecules constantly collide with thebigger particles embedded in them, at any moment there is imbalance inthe net force impacted by these small molecules on the bigger particles.This imbalance in the net force results in the movement of the biggerparticles and results in the net force being random in direction andmagnitude. Therefore, the imbalance in the net force causes randommovement of the bigger particles. It follows that in order for thisrandom motion to be detectable macroscopically, for example, either withnaked eye or with a simple optical microscope, the size of the biggerparticles cannot be too big compared to the size of the small molecules.The Brownian motion of particles in three dimensions is well describedby a simple equation for mean-square displacement (MSD):

δx ²=6Dt

where t is time. The MSD used in Statistical mechanical description ofparticle motion is defined as:

{x(t)−x(0)}²

where x(t) represents the position of the particle at time t, x(0)initial position and the angled brackets represent time average.Supposing one knows the positions of a particle over a time duration atan interval δt, then MSD can be calculated from the equation:

δx ²=[(x(t ₁)−x(0))²+(x(t ₂)−x(0))²+(x(t ₃)−x(0))²+ . . . +(x(t_(n))−x(0))² ]/n

Where n is the number of consecutive positions of the particle.

The only constant in this equation that characterizes the particle isthe diffusion constant, D. The diffusion constant D can be calculatedfrom Einstein's relation

D=μk _(B) T

where k_(B) is the Boltzmann constant, μ is the mobility of thediffusing particle, and T is the temperature of the medium (liquid) theparticle is diffusing in.

For a spherical particle, the mobility can be calculated from Stokes lawfor the drag force on the particle and Einstein's relation simplifies to

$D = \frac{k_{B}T}{6{\pi\eta}\; r}$

where ‘r’ is the radius of the particle, η is the dynamic viscosity ofthe medium (liquid). In addition to the simple equation that can be usedto calculate the diffusion coefficient D of the particle, experimentallymeasured values are also widely available for polymer micro-beads andDNA molecules. So given the temperature T and the nature of the medium(e.g., the dynamic viscosity η) in which the particle is present, themean-square displacement (δx²) of the particle can be calculated andwill be linear in time duration of the observation of the particle.

In the case of experiments tracking particle motion either bycellular-phone based microscopes or research microscopes with highnumerical aperture, since the particle position is detected only in atwo-dimensional plane, the equation for MSD corresponds totwo-dimensional diffusion which is δx²=4Dt. Because of this differencein the coefficients, the diffusion constant for two-dimensionaldiffusion will be larger than for three-dimensional diffusion.

FIG. 1 illustrates a sample surface 10 with a liquid 20 on the samplesurface 10 according to an embodiment. An example micron size bead 30 isin the liquid 20. The liquid 20 is made up of small molecules 40. Themicron size bead 30 is representative of the bigger particle in theliquid of the small molecules 40. As discussed above, the smallmolecules 40 bombard the bead 30 and exert a net force on the biggerbead 30. The bigger bead 30 travels in a random direction according toBrownian motion and the random direction previously traveled by thebigger bead 30 is shown by a displacement trace/line 50. FIG. 1 shows anexample with a single micron bead 30 for ease of understanding, but itshould be appreciated that the same analogy applies for numerous beads30 in the liquid 20.

Detection of Specific DNA/RNA Sequences

The mean-square displacement (δx²) of a free Brownian particle (such asthe bead 30) keeps increasing linearly with time as was just discussed.However, if the particle is tethered at one end to the surface of thesample container, then the particle's motion will be restricted by thetether length according to embodiments, as shown in FIG. 2. This effectcan be exploited to detect the presence of a DNA sequence in thefollowing way.

FIG. 2 is a cross-sectional view of a system 200 utilized for detectionof a specific DNA sequence in an unknown DNA molecule 210 being testedaccording to an embodiment. For the sake of clarity and simplicity, thesmall molecules 40 of the liquid 20 are not shown in subsequent figures.Also, for explanation purposes, only two beads 30 are illustrated but itshould be appreciated that numerous micron size beads (e.g., 100-1000)may be utilized in embodiments.

In FIG. 2, the unknown DNA molecule 210 has a nucleic acid sequence thatneeds to be detected, and the unknown DNA molecule 210 is the moleculebeing tested. The unknown DNA molecule 210 may be the DNA/RNA of avirus, pathogen, a food product, etc., which needs to be tested. Theunknown DNA molecule 210 is to be tested against a short segment of itscomplementary sequence. This segment can be of 8 to 40 bases long andchosen such that the double-stranded DNA that it forms with the unknownDNA molecule 210 will have sufficiently higher melting temperature thanthe temperature at which the experiments are carried out. The meltingpoints of such segments can be calculated by methods based onnearest-neighbor interactions of the nucleic acid bases described byother researchers and their later improvements. (John SantaLucia Jr.(1998). “A unified view of polymer, dumbbell, and oligonucleotide DNAnearest-neighbor thermodynamics”. Proc. Natl. Acad. Sci. USA 95 (4):1460-5. doi:10.1073/pnas.95.4.1460; Breslauer, K. J.; Frank, R; Blocker,H; Marky, L A et al. (1986). “Predicting DNA Duplex Stability from theBase Sequence”. Proc. Natl. Acad. Sci. USA. 83 (11): 3746-3750.doi:10.1073/pnas.83.11.3746). They are also available as open-accesssoftware tools such as Oligo Solver 3.0 (Integrated DNA Technologies,http://www.idtdna.com/calc/analyzer). While designing these shortsegments their tendency to form partial double-strand DNA segments dueto self-hybridization can be calculated using DINA Melt web server,http://mfold.rna.albany.edu/?q=DINAMelt/Two-state-folding. It makescomputational folding studies of the possible self-hybridization pairsand calculates the melting temperatures of such configurations. Keepingin mind both the high melting temperature of the double-strand segmentformed with the unknown DNA molecule and minimal tendency forself-hybridization the sequence of the short segment DNA can bedesigned. As such, the unknown DNA molecules 210 are the sample beingtested in the liquid 20.

The DNA molecule 210, having a sequence that needs to be detected, canbe attached to the beads 30 at one end by using beads 30 coated with afirst molecule 215 represented as diagonal lines. The molecule 215 ischosen to attach to the material of the beads 30. In one implementationto attach the first molecule 215 to the beads 30, the beads 30 may bepolystyrene beads and the coated molecule 215 may be the protein(molecule) streptavidin. The protein streptavidin is coated on and bindsto the beads 30.

In another implementation, the material of the beads 30 may bepoly-methyl-methacrylate (PMMA) beads in the size range of 0.2 to 2.0microns such as those available from any of these commercial vendors:Bangs Labs, Life Technologies and PolySciences and the first molecule215 may be streptavidin protein molecules, such that the first molecule215 binds to the beads 30.

In yet another implementation, the material of the beads 30 may besilica (SiO₂) beads in the size range of 0.2 to 2.0 microns such asthose available from any of these commercial vendors: Bangs Labs, LifeTechnologies and PolySciences and the first molecule 215 may bestreptavidin protein molecules, such that the first molecule 215 bindsto the beads 30.

In order to facilitate the DNA molecule 210 attaching to the bead 30coated with molecule 215, the end of the DNA molecule 210 is attachedwith a second molecule 220 that is designed/chosen to attach to thefirst molecule 215 (e.g., streptavidin). The second molecule 220 has adual purpose, which is 1) to bind to the unknown DNA molecule 210 and 2)to bind to the first molecule 215. Similarly, the first molecule 215 hasa dual purpose, which is 1) to bind to the bead 30 and 2) to bind to thesecond molecule 220.

In one implementation, the second molecule 220 may be a biotin moleculethat will bind to the streptavidin molecules 215 (i.e., first molecules215) coated on the bead surface of bead 30. Accordingly, this(combination of the first molecule 215 and second molecule 220) formsDNA molecules 210 attached at one end to the bead surface 30.

In FIG. 2, the sample surface 10 (such as, e.g., a microscope glassslide after some pre-cleaning if necessary) is coated with antibodies202 that will bind to Digoxigenin molecules 203. The short probe DNAmolecules 205 are attached with Digoxigenin molecules 203 on one end(could be short probe RNA molecules when testing an unknown RNA moleculeor RNA molecule attached with short probe DNA molecule). The short probeDNA molecules 205 contain a segment of the complementary sequence for aknown DNA/RNA molecule, which means that the short probe DNA molecules205 (complementary sequence) is expected/known in advance to attach tothe known DNA molecule (e.g., a known virus being tested). Therefore, ifthe free end (the end not attached to the bead surface 30) of theunknown DNA molecule 210 binds to the short probe DNA molecules 205(i.e., complementary sequence of a segment of the known molecule), theunknown DNA molecule 210 is the same as the known DNA molecule. Becauseby choice, the sequence of the short probe DNA molecule 205 is chosensuch that to a very high degree it (the sequence of the short probe DNAmolecule 205) belongs only to the known DNA molecule. The converse isalso true. If the free end (the end not attached to the bead surface 30)of the unknown DNA molecule 210 does not bind to the short probe DNAmolecules 205 (i.e., complementary sequence of a segment of the knownmolecule), the unknown DNA molecule 210 is not the same as the known DNAmolecule.

As the DNA-attached bead 30 comes in contact with this short probe DNAmolecule 205, the unknown DNA molecules 210 form the tether therebyrestricting the Brownian motion of the DNA-attached bead 30. Therefore,the presence of a specific DNA sequence (in the unknown DNA molecule210) of the DNA-attached bead 30 can be detected (indirectly).

The short probe DNA molecules 205 are known and may be the DNA moleculesof the virus, pathogen, food product, etc., that is being tested for.Since the short probe DNA molecules 205 are the complementary sequenceof what the unknown DNA molecule 210 should be, the short probe DNAmolecules 205 are designed to bind to the expected (but unknown)sequence of the DNA molecule 210. When the DNA molecules 210 are thecomplementary sequence to the short probe DNA molecules 205, the two DNAmolecules 205 and 210 bind to form a double strand DNA molecule. In aDNA double helix, each type of nucleobase on one strand binds with justone type of nucleobase on the other strand. This is called Watson-Crickor complementary base pairing. The short probe DNA molecules 205 aresingle strand DNA molecules, while the tested DNA molecules 210 aresingle strand DNA molecules. One skilled in the art understands how toprepare complementary DNA sequences, single strand DNA, and short DNAprobes.

In one embodiment, single-stranded DNA molecules 210 made of the sametype of bases (A, T, G or C) can be used for detecting their binding tothe beads 30 and the resulting restricted Brownian motion. Thesehomopolymer DNA molecules can be custom-made with a pre-attachedDigoxigenin molecule at one end and the biotin molecule at the otherend. Here is one example of such a DNA molecule:Digoxigenin-C6-amino-(TTTTTTTTTT)₁₅-3′-biotin. These moleculesconsisting of 150 Thymine bases can be obtained from commercial vendors(Midland Certified Reagent Company Inc™, Midland, Tex. and IntegratedDNA Technologies, Inc™, Coralville, Iowa). These DNA molecules 210 canbe attached to the beads 30 coated with streptavidin molecules throughbiotin molecules 220 in one end of them. The DNA-attached beads will befilled in the sample chamber to test their binding to the antibodymolecules coated on the glass surface of the sample cell through theDigoxigenin molecules at the other end of the DNA molecules.

In another embodiment, long double-stranded DNA molecule frombacteriophage viruses can be used as DNA molecules 210. The linearlamda-DNA extracted which has 48,502 bases can be used (available fromNew England Biolabs, Inc™, Ipswich, Mass.). It has two cohesive orsticky overhangs each 12 bases long at each end. Their sequences are:5′-GGGC GGCG ACCT-3′ and 5′-AGGT CGCC GCCC-3′. The short DNA oligomers5′-GGGC GGCG ACCT-3′-biotin and 5′-AGGT CGCC GCCC-3′-Digoxigenin can bemixed with Lamda DNA one at a time so that one of these DNA oligomerswill bind to their sticky ends (can be purchased from Integrated DNATechnologies, Inc™). After attaching one of them they will be ligated tochemically link to the long strands of Lamda DNA to which they attach bycomplementary pairing. After ligation, for example, the oligomer withthe biotin on one end can be mixed with streptavidin coated beads insolution. The DNA attached beads can be isolated by sedimenting thebeads by centrifugation and extracting the supernatant solution. Theseisolated DNA attached beads can be mixed with the Digoxigenin attachedoligomers to bind to the other overhang in the Lamda DNA and ligated. Atthe end, one will have Lamda DNA with Digoxigenin ends that are free tobind to the antibody coated surface.

In yet another embodiment, a single-stranded DNA molecule M13mp18 from avirus can be utilized as DNA molecules 210. This DNA has 7249 bases andits contour length is about 5 microns which is a sufficiently longtether for the bead. The DNA sequence CTC GCG CCC CAA occurs near the 5′end of this DNA molecule and has a melting temperature of 51.9 C whichis much higher than normal operating temperature. The DNA sequence ATGCTC TGA GGC TTT TA occurs near the 3′ end and has a melting temperatureof 47.3 C much higher than normal operating temperature. Therefore thesetwo sequences can be utilized to design short DNA oligomers attachedwith biotin and Digoxigenin molecules. The DNA oligomer, Biotin-3′-GAGCGC GGG GTT-5′ will form complementary base pairing with the sequencenear the 5′ end, namely, CTC GCG CCC CAA. The biotin attached to the 3′end will bind to the Streptavidin molecules coated on the beads 30 whenmixed with them in solution. In order to break any unwanted hair pinformation in these DNA oligomers the mixture with the beads can beheated above their complementary base pair melting temperature andslowly cooled across their melting zone and then cooled rapidly toeither room temperature or quenched in an ice-bath. The other shortoligomer 3′-TAC GAG ACT CCG AAA AT-5′-Digoxigenin can be mixed with theDNA attached beads and will bind to the antibody coated on the samplecell surface thereby forming DNA tethers for the beads 30. The dynamicsof binding of either biotin to streptavidin beads or the Digoxigenin toantibody molecules coated on the sample cell surface can be made fasterby using a homopolymer spacer between biotin or Digoxigenin and theshort oligomer DNA sequence. The length of such homopolymers can be inthe range of 20 to 200 bases long. By having the single-strand DNA beingmuch more flexible than double-stranded DNA (persistence length fordsDNA is 50 nm and for ssDNA is ˜2 nm which is twenty five timesshorter), these homopolymers will let the binding molecules Biotin orDigoxigenin to explore different configurations before they bind totheir counterparts (Streptavidin or Antibody molecules as discussedbefore) accelerating their time for binding.

FIG. 2 shows the DNA molecule 210 attached/bound to the short probe DNAmolecule 205, which indicates that the DNA molecule 210 and the shortprobe DNA molecule 205 are the same molecule. In other words, this meansthat targeted DNA molecule 210 and the short probe DNA molecule 205 areboth from the known molecule (although they may have complimentarynucleic acid sequences). To recognize that the DNA molecule 210 isattached to the short probe DNA molecule 205, the random motion of thebeads 30 is monitored. The displacement of untethered beads 30 would besimilar to the free Brownian motion of the displacement trace 50 shownin FIG. 1 which does not restrict the space the bead can explore overtime. However, when the DNA molecule 210 is attached to both the bead 30and the short probe DNA molecule 205 (i.e., beads 30 are tethered to thesample surface 10 via the DNA molecule 210 being tested), the bead 30 isrestricted to (only) moving to the dashed bead locations 30A and 30B. Asone depiction in a two-dimensional space, the tethered beads 30 can movea maximum distance of D1 along the x-axis in one direction, and thetethered beads 30 can move a maximum distance of D2 along the x-axis inthe opposite direction. The extent of the movement for the tetheredbeads 30 in the x-axis is limited to D0=D1+D2, which is based on thelength of the DNA molecule 210 (i.e., the tether). By having the bead 30tethered by the DNA molecule 210, the potential motion, which islimited/restricted Brownian motion of the tethered bead 30, is roughlylimited to the volume of a sphere along the x, y, and z axes. FIG. 3A isa top-down view of the system 200 utilized for detection of a specificDNA sequence in the unknown DNA molecule 210 being tested according toan embodiment. FIG. 3A shows that each tethered bead 30 (i.e., when theDNA molecule 210 is attached to both the bead 30 and the short probe DNAmolecule 205) may have the limited range of Brownian motion that formsand/or is confined to a circular shape 305 (within the two-dimensionalspace). The diameter of the circular shape 305 is D0 where D0=D1+D2 asshown in FIG. 2. This limited and restricted Brownian motion, confinedto the area of the circular shape 305 (which is a sphere in athree-dimensional space), of the bead 30 tethered to the sample surface10 (i.e., the short probe DNA molecules 205) by the DNA molecule 210 isdetermined to be positive identification of the known molecule. In thecase when more than one DNA molecule 210 is attached to the bead 30, themotion of the bead can be further restricted to a circular shape withthe diameter significantly smaller than D0=D1+D2. This is also apositive identification of the known molecule.

This limited/restricted Brownian motion of the tethered beads 30 can beoptically recognized and traced via a cellular phone 401, a chargecoupled device (CCD), and/or a microscope. When the limited/restrictedBrownian motion of the tethered beads 30 is recognized, it is determinedthat the tested DNA molecule 210 is the same molecule as the short probeDNA molecule 205. The identification of the short probe DNA molecules205 are known in advance.

For the sake of clarity and simplicity, the small molecules 40 of theliquid 20, the short probe DNA molecules 205, and the DNA molecules 210are omitted in FIG. 3A. Also, for explanation purposes, only a singlebead 30 is illustrated but it should be appreciated that numerous micronsize beads (e.g., 100-1000) may be utilized.

In the case when the unknown DNA molecules 210 do not bind with theshort probe DNA molecules 205, this means that the sequence of theunknown DNA molecule 210 is not the complementary sequence to the shortprobe DNA molecules 205. Therefore, the unknown DNA molecules 210 arenot the same molecule as the known short probe DNA molecules 205, andthe unknown DNA molecules 210 can be ruled out as being the samemolecule as the known molecule (e.g., a particular virus).

FIG. 3B is graph illustrating the effects of DNA tethering on x and ybead displacement according to an embodiment. In FIG. 3B, waveform 306illustrates the distribution of the bead x- or y-position displacementsdue to the free Brownian motion in the absence of tethering by thetargeted nucleic acid sequence in the sample. In contrast, waveform 307shows a much narrower distribution of the bead displacement whentethering is formed by the targeted nucleic acid sequence in the sample.

FIG. 3C is a graph illustrating the effects of DNA tetheringmean-squared displacement according to an embodiment. Waveform 308illustrates the mean-squared displacement of the free beads in twodimensions of their Brownian motion showing that the free beads exhibitlinear behavior obeys δx²=4Dt where D is the diffusion constant.

In contrast, plot 309 illustrate that, when tethering is formed by thetargeted nucleic acid sequence, the tethered beads' motion will beslowed down for smaller displacements resulting in smaller diffusionconstant over a shorter time duration. The tethered beads may alsoexhibit non-linear behavior arising from the polymer elasticity of thetether. Plot 310 illustrates that, on a longer time duration as thebeads try to move beyond the length of the tether, they become confinedto a circle. This results in the flattening of their mean-squareddisplacement.

According to another embodiment, FIG. 13 is a cross-sectional view of asystem utilized for detection of a specific DNA sequence within anunknown DNA molecule being tested. FIG. 13 is similar to FIG. 2 exceptthat the second molecule 220 is removed. In this embodiment, one end ofthe unknown DNA molecule 210 is attached directly to the first molecule215 (i.e., attached to the beads 30) without the second molecule 220.

According to another embodiment, FIG. 14 is a cross-sectional view of asystem utilized for detection of a specific DNA sequence within anunknown DNA molecule being tested. FIG. 14 is similar to FIG. 2 exceptthat the second molecule 220 is removed and an additional short probeDNA molecule 1405 is added. In this embodiment, the additional shortprobe DNA molecule 1405 is attached to the first molecule 215 (i.e., theadditional short probe DNA molecules 1405 are attached to the beads 30).Accordingly, one end of the unknown DNA molecule 210 is attached to theadditional short probe DNA molecule 1405. In one implementation, theadditional short probe DNA molecule 1405 may be the same nucleic acidsequence as the short probe DNA molecule 205.

Optical Detection of Bead Motion with a Cellular Phone

Many smart phones (and tablets) available today in the market likeiPhones® (4S, 5, 5C and 6), Samsung® Galaxy, HTC™ and other Android™phones have a good camera with video recording capabilities. However,their current minimum-focusable distance and the magnificationachievable at that distance are not adequate to detect the presence ofmicron to submicron (10⁻⁶ to 10⁻⁵ meter) beads 30 in the solution. ForiPhone® 5, the minimum-focusable distance is about 5 centimeters (cm)from the front of the camera, and the magnification achieved is aboutfew times which directly impacts the resolution. Therefore, a speciallens adapter is needed for current camera phones not having adequatemagnification and resolution, and the special lens adapter (e.g., balllens holder assembly 400 shown in FIGS. 4 and 5) can be attached tothese cellphone cameras. It is noted that newer phones or electroniccommunication devices may be designed with camera technology that hashigher magnification and resolution, and some embodiments may notrequire the special lens adapter.

The special lens adapter has special characteristics like a short focallength and high numerical aperture that can result in magnification inthe range of 100-200 times (100-200×) and a spatial resolution of 1 to 2micrometers. Such characteristics can be achieved by a ball-lens basedadapter (e.g., ball lens holder assembly 400 shown in FIGS. 4 and 5),and it can be attached to and/or placed in front of the camera of thesecellular phones in a special holder (e.g., micropositioner 418 in FIGS.4 and 5). In one embodiment, a ball-lens of diameter 3.0 mm made ofglass (refractive index 1.52) with very low surface roughness (withinmicrons) placed in a holder and kept over the sample to be imaged wasfound to have a magnification of approximately 180×, and a resolution ofabout 1 micron which was adequate to image the beads 30 under suitablelow-light illumination conditions (FIGS. 4 and 5).

FIG. 4 is a system 480 depicting a cellular phone based bead motionimaging microscope according to one embodiment. In this experimentalconfiguration, the ball lens holder assembly 400 is placed over thesample chamber in the glass slide 10 covered by a glass cover slip 409either with the help of spacers 407 and 413 (in one implementation) orwithout spacers 407 and 413 (in another implementation). The ball lensholder assembly 400 is designed such that the bead sample solution 20below the cover slip 409 is in the focal plane of a ball lens 405. Thecellular phone camera's horizontal position is adjusted to be in thecenter of the field of view of the spherical ball lens 405 with amicropositioner 418 and the vertical position adjusted to form the imageof the beads 30 in the solution 20.

A cellular phone 401 includes a camera 450 with an electronic imagesensor 402 (can also be other imaging sensors (including CCDs) withfocusing optics). The electronic image sensing surface 402 is positionedbehind a camera focusing lens assembly 403. The cellular phone 401includes a processor, memory (including removable memory), cameracomputer-executable instructions stored in memory, video recordingcomputer-executable instructions stored in memory, antenna, transceiver,power source (e.g., battery), user interface (touch screen, key pad,voice activation) etc., as understood by one skilled in the art.

The ball lens holder assembly 400 includes a resolution limitingaperture 404. An emission filter (not shown), that blocks the excitationlight in the case of imaging fluorescent beads or fluorescentbiomolecules like DNA and proteins, can be placed over resolutionlimiting aperture 404 in one implementation.

The ball lens holder assembly 400 includes the spherical ball lens 405made from glass, e.g., with a diameter 3.0 millimeters (mm) positionedin a lens holder 430. In another implementation, other smaller or largerdiameters such as, e.g., 2.5, 4.0, and 5.0 mm may also be used as thespherical ball lens 405 with the appropriate lens holder assembly. Thelens holder 430 may have a tapered opening 406 to accept a light cone oflarge angle from the sample plane formed by the solution 20 of beads 30.Spacers 407 and 413 keep the required separation between the ball lensholder assembly 400 and the glass slide 10 that contains the beadsolution 20. Double-sided tape or adhesive may act as spacers 408 and411 for the sealed sample chamber 435. In another implementation, asample cell may also consist of a glass or transparent polymer-basedmicrofluidic cells. A glass cover slip 409 acts as the lid for thesample chamber 435. The sealed sample chamber 435 comprises the glassslide 10 and glass cover slip 409. The bead solution 20 (containing thebeads 30) that is being imaged (via camera 450 of the cellular phone401) is placed on the glass slide 10. The glass slide 10 forms thebottom surface of sample chamber 435 enabling different kinds offunctionalization such as with antibody and protein coatings to bind toeither the DNA molecule 210 or other small molecules 220 like biotin.

The ball lens holder assembly 400 works with light limiting apertures414 and 416. An excitation filter (not shown) can be placed over theaperture 414 for imaging fluorescent beads and fluorescent DNA and RNAbiomolecules. A mirror 415 is utilized for reflecting light towards thesample chamber 435 and the ball lens holder assembly 400.

A battery-operated LED flash lamp, with white light or colored light,may be utilized as a light source 417. As another option, the lightsource 417 may be a single LED suitably wired for electrical power. Thelight source 417 may be a small laser diode suitably powered. In thecase of a single LED or laser diode, the light source 417 can be placeddirectly under the aperture 414 without the need for the mirror 415 andthe light limiting aperture 416. The light source 417 may also be formedwith a flat LED chip mounted on a suitable holder, wired for electricalpower, and placed under the aperture 414. A micropositioner 418 holdsthe cellular phone 401 and can move in three orthogonal x, y, and zdirections. The micropositioner 418 is used for positioning the cellularphone camera 450 directly over the lens holder assembly 400 centered atthe resolution limiting aperture 414 and for adjusting the verticaldistance from this aperture 414 to get a good focus of the beads in thesolution.

FIG. 5 is a system 580 depicting a cellular phone based bead motionimaging microscope according to another embodiment. The system 580 inFIG. 5 includes the features of the system 480 in FIG. 4. However, inthis experimental configuration, the ball lens holder assembly 400 isattached to the cellular phone 401 with the field of view of thespherical ball lens 405 approximately centered with the field of view ofthe cellular phone camera 450. The alignment can be done by adjustingthe lens holder assembly 400 to image a bright uniformly lit object or alight source such as an LED flash lamp in order to ensure that the imagehas uniform intensity. The horizontal and vertical positions of thecellular phone 401 with the lens assembly 400 are adjusted with themicropositioner 418 to bring the beads 30 in the solution 20 into focus.Once aligned, the cellular phone 401 may be attached to the ball holderassembly 400 with an attaching piece 505, and the attaching piece 505may be clamps, adhesive material, etc. In addition, the use of the imageresolution limiting aperture 404 is optional in FIG. 5, and thus may notbe utilized in one implementation.

In the experimental setups described in FIGS. 4 and 5, a single balllens 405 has been used to image the Brownian motion of the beads 30 insolution 20. Depending on the planarity of the glass slide samplechamber 435 and the slight unintended off-centering of the ball lensfield-of-view with respect to the cellular phone camera field-of-view,some distortion of the bead images near the edge of the field of viewmight occur. This distortion can be reduced substantially or removed iftwo half-ball lenses are used. This configuration also allows for aslightly wider field-of-view than achievable with a single ball lens, asillustrated in FIG. 6.

FIG. 6 illustrates a Wollaston-like doublet arrangement 600 that may beutilized according to an embodiment. The two half-ball lenses 601 and603 are arranged in such a way that their curved surfaces face eachother. Aperture 404 is the resolution limiting aperture. Half-ball lens602 is the smaller half-ball lens that can be of diameter 1.5 to 2.5 mm.The connecting light path hole 603 acts as another resolution limitingaperture. Half-ball lens 604 is the bigger half-ball lens which can havediameter larger than the smaller half-ball lens by 1.0 to 2.5 mm.Wollaston-like doublet arrangement 600 has a field aperture 605. Whilethe aperture 605 should be larger to accept a large cone of lightscattered from the sample material, the apertures 603 and 404 can beoptimized to achieve a spatial resolution of 1 to 2 microns. Thevertical separation between the two lenses can be optimized to achieve atotal magnification of 150 to 200 times for observing the motion of 1micron sized beads. Wollaston-like doublet arrangement 600 can beutilized in place of the elements 405 and 406 in the ball lens holderassembly 400.

Example Experimental Data 1

Before detecting Brownian motion of the beads 30, experiments werecarried out imaging beads that are stuck to a glass surface or frozen ina gel matrix coated on a glass slide in order to compare with therecordings of moving beads 30. Images recorded with 1.2 micron sizedbeads 30 are shown in a raw image 750 and processed image 755 in FIGS.7A and 7B, respectively.

FIG. 7A illustrates image 750 showing polystyrene beads of average size1.2 microns stuck on a glass slide. The beads were obtained from BangsLaboratories, Inc™, Fishers, Ind. Using the setup described in FIG. 5,the original image 750 of the beads stuck on the glass slide is recordedwith transmitted light from an LED flash white light illumination. Theindividual beads can be seen as bright spots surrounded by darkercircles 701.

FIG. 7B illustrates image 755 pre-processed after converting to agray-scale image and band pass filtering for optimal featureenhancement. The circles 702 are centered around bead positions inframe-1 while the crosses are from bead positions in frame-2, whereframe-1 and frame-2 are consecutive in a video clip. The bead positionsdetected after pre-processing the original image 750 by band-passfiltering with intensity threshold setting are shown as the circles 702.In FIG. 7B, a large number of detected bead positions can be seen.

For comparison, the bead positions detected with a MATLAB® functioncalled imfindcircles function is shown in FIG. 7C, and the MATLAB®function has a large number of beads detected (circles 703). FIG. 7Cillustrates image 760 showing bright spots identified by imfindcirclesfunction in MATLAB® after some pre-processing. The particular circle 703is drawn to the size of the radius found by the imfindcircles functionfor each corresponding bead 30 location in the image.

FIG. 7D illustrates a histogram graph 765 showing the distribution ofthe radius of the detected circles 703 in pixels from the image 760 inFIG. 7C. For the image 760, bead positions corresponding to circles 703were identified (using MATLAB®) in the first frame of a video clip andtheir corresponding positions were identified in the second frame givingallowance for a maximum displacement of 10 pixels between these twoframes. However, the analysis shows that the dominant amount ofdisplacement of beads between these two frames is less than 0.05 pixels,and it further shows that the larger displacements that are still below1 pixel are negligible as shown in FIGS. 7E-1 and 7E-2 and FIGS. 7F-1and 7F-2.

It is noted that the displacement of a bead is the length/distance thata bead moves in a particular direction, along the x-axis or y-axis.Since the microscope images the motion in a plane which is twodimensional, in general, the bead displacement will be in an arbitrarydirection in the plane. For simplification of the analysis, only x- ory-component of the bead motion was analyzed which corresponds todiffusion in one dimension. However, from the x- and y-positions of thebeads obtained in the analysis their total displacement,

δ{right arrow over (r)}={right arrow over (r ₂)}−{right arrow over (r₁)}=({right arrow over (x ₂)}−{right arrow over (x ₁)})+({right arrowover (y ₂)}−{right arrow over (y ₁)})=δx·{circumflex over (x)}+δy·ŷ

Where {right arrow over (r₂)} and {right arrow over (r₁)} denote the 2Dposition vector of the bead displacements, {circumflex over (x)}, ŷrepresent the unit vectors along x- and y-directions respectively.Therefore, square of radial displacement can be calculated as:

δ{right arrow over (r)} ² =δx ² +δy ²

and the mean-squared displacement δr² can be calculated from thesuccessive x- and y-positions of the beads and it obeys the followingequation:

δr ²=4Dt

FIGS. 7E-1 and 7E-2 illustrate the displacement in bead positionsbetween two consecutive images. FIGS. 7E-1 and 7E-2 are histogram graphs770 and 775, respectively, of the distribution in bead positions betweentwo consecutive video frames (which included the image 760). Thehistogram graph 770 shows that the smaller displacement is less than0.05 pixels for the x-position, and most of the displacements are within−0.005 in FIG. 7E-1.

FIG. 7E-2 shows a similar behavior for y-positions in the histogramgraph 775. This analysis shows both the robustness of the experimentalsetup in FIG. 5 (as well as FIG. 4) keeping the residual mechanicalvibrations from the floor low enough for observing the static images ofthe beads and the effectiveness of the algorithm in finding the beadpositions from the image frames of the video clip frames.

FIGS. 7F-1 and 7F-2 respectively illustrate the x and y displacements(both small and large). FIG. 7F-1 shows a histogram graph 780 of thedistribution of total displacement in bead positions between twoconsecutive video frames. The histogram graph 780 shows that thedisplacement less than 0.05 pixels for x-positions dominates the totaldisplacement. Similarly, histogram graph 785 shows that the displacementless than 0.05 pixels for y-positions dominates the total displacement.

On the other hand, the bead displacements between first and third framesspanning 0.5 pixels are still smaller than a single pixel as shown inFIGS. 7G-1 and 7G-2. FIG. 7G-1 shows histogram graph 790 ofx-displacement while FIG. 7G-2 shows histogram graph 795 ofy-displacement. Note that most of the x and y displacements of the beadsin frame-1 and frame-3 are within 0.5 pixel which is good and testifiesof the high accuracy of these algorithms in finding the bead centerpositions with sub-pixel accuracy. This shows the adequacy of both theexperimental setup in FIG. 5 (along with FIG. 4) and the algorithms forrecording and locating the bead positions with sub-pixel accuracy.

An example algorithm for detecting and tracking the movement (i.e., beadpositions) of the individual beads 30 is provided below according to anembodiment. For a video clip/recording of the motion of the beads 30(e.g., captured with the cellular phone 401), the example algorithm maybe utilized to detect and track beads from one video frame to the next(consecutive video frame). The algorithm may be executed by and storedin memory of the cellular phone 401 and/or the cellular phone 401 maysend the recorded video clip/recording to a computer system (such as thecomputer system 1200) for analysis; the computer system has memory tostore the algorithm. The example algorithm is configured to identifyeach circular bead and store each bead's pixel location in a videoframe, such that the algorithm identifies and stores each bead's pixellocation throughout consecutive video frames. For example, the algorithmis configured to track and identify each bead's pixel location overvideo frames (e.g., over consecutive video frames). The algorithm isconfigured to measure the x-displacement and y-displacement of each bead30 from one video frame to the next video frame, such that the algorithmstores the change in pixel location for each bead. The algorithmdetermines an x-displacement and y-displacement for each bead 30 in thex-axis and y-axis, over a predefined number of video frames (i.e., aparticular time, such as, e.g., 1, 3, 5, 10, 15, 20, 25 . . . 60seconds). For the particular sample having the unknown DNA molecule 210being tested, the algorithm determines the total x-displacement for allof the individual beads 30 and the total y-displacement for all of theindividual beads 30. The algorithm is configured to compare the totalx-displacement and y-displacement for the beads 30 being tested with theunknown DNA molecule 210 attached (i.e., corresponding to the Brownianmotion of the tested DNA molecule 210) to the previously stored totalx-displacement and y-displacement for the same size beads without theunknown DNA molecules 210 attached (i.e., corresponding to free Brownianmotion). When the total x-displacement and y-displacement for the beads30 being tested with the unknown DNA molecule 210 attached (i.e.,corresponding to the Brownian motion of the tested DNA molecule 210) isless than (by a predetermined amount) the previously stored totalx-displacement and y-displacement for the same size beads without theunknown DNA molecules 210 attached (i.e., corresponding to free Brownianmotion), the algorithm determines that the Brownian motion is restricted(i.e., the beads 30 are tethered to the sample surface 10 via the DNAmolecule 210) and the unknown DNA molecule 210 contains the knownspecific nucleic acid sequence being tested for.

However, when the total x-displacement and y-displacement for the beads30 being tested with the unknown DNA molecule 210 attached (i.e.,corresponding to the Brownian motion of the tested DNA molecule 210) isabout equal to (within a predetermined margin) the previously storedtotal x-displacement and y-displacement for the same size beads withoutthe unknown DNA molecules 210 attached (i.e., corresponding to freeBrownian motion), the algorithm determines that the Brownian motion isnot restricted (i.e., the beads 30 are not tethered to the samplesurface 10 via the DNA molecule 210) and the unknown DNA molecule 210does not contain the known specific nucleic acid sequence being testedfor.

Example Experimental Data 2

Referring to FIGS. 8A-1 and 8A-2, a similar analysis was carried outwith micron sized beads affixed in a gel matrix coated on a glass slide(from PolySciences Inc., of Warrington, Pa.). FIGS. 8A-1 and 8A-2respectively show the fluorescent image 805 recorded from these polymerbeads and the processed image 810 with the bead positions identified intwo consecutive frames. The images are extracted from the video cliprecorded from 6 micron size calibration standard beads (fromPolySciences, Inc., Warrington, Pa.).

In FIG. 8A-1, the original fluorescent image 805 was recorded withtransmitted light from LED flash light illumination using the setupdescribed in FIG. 5 (although the setup in FIG. 4 could be used) butwith an excitation filter placed over the aperture after the mirror. Thecircle 801 shows individual beads in the original fluorescent image 805.In FIG. 8A-2, circles 802 are centered around bead positions in frame-1and the crosses are centered at bead positions in frame-2 extracted froma video clip. The algorithm first identifies the bead position at acoarse level which is used to find the bead position to sub-pixelaccuracy by analyzing intensity distribution around this position over a10×10 to 20×20 window. The window is user controllable. Intensitydistribution around one such peak is seen in FIG. 8B.

FIG. 8B is an graph 815 of pixel-level intensity distribution of adetected bead image showing the extent of the bright region spanningabout 10×10 pixels. The displacement of pixels in graph 815 is muchlarger than the largest displacement 0.05 pixels obtained from theanalysis of the images in FIGS. 8D-1, 8D-2, 8E-1, and 8E-2.

FIG. 8C-1 illustrates an image 820 showing bright spots identified bythe imfindcircles function in MATLAB®, and a large number of beadpositions are identified. Each circle 803 is drawn to the size of theradius found by imfindcircles function for each bead position. FIG. 8C-2is a graph 825 illustrating the distribution of found circle radii inpixels.

Analysis of the displacement of beads in two consecutive frames showsthat the displacement less than 0.01 pixels dominates the distributionin FIGS. 8D-1, 8D-2, 8E-1, and 8E-2. This is further consolidation ofthe adequacy of the experimental setup and the algorithms for locatingthe bead positions with sub-pixel accuracy.

FIGS. 8D-1 and 8D-2 illustrate displacement in bead positions betweentwo consecutive images of a video clip. FIG. 8D-1 is a histogram graph830 of the distribution of total displacement in bead positions betweentwo consecutive video frames, and shows that a displacement less than0.01 pixels for x-positions dominates the total displacement. FIG. 8D-2is a histogram graph 835 of the distribution of total displacement inbead positions between the two consecutive video frames, and also showsthat a displacement less than 0.01 pixels for y-positions dominates thetotal displacement. Note that the analysis of 1.2 micron beads stuck onglass slide also showed similar behavior in FIGS. 7F-1 and 7F-2, whichconsolidates the effectiveness of the algorithm for finding the positionof small as well as large beads.

FIGS. 8E-1 and 8E-2 illustrate x and y displacements (both small andlarge). FIG. 8E-1 is a histogram graph 840 of the distribution of totalx-displacement in bead positions between two consecutive video framesshowing larger displacements as well. However, the smaller displacementsare peaked at close to zero for x-displacement.

FIG. 8E-2 is a histogram graph 845 of the distribution of totaly-displacement in bead positions between two consecutive video frames.The y-displacement in graph 845 has similar behavior the x-displacementin graph 840.

Example Experimental Data 3

Now, turning to another experiment, dilute solutions of polystyrenebeads of 1.2 micron size were used to record the motion of the beads. Animage 905 of a frame from the video clip recording the bead motion isshown in FIG. 9A. The individual beads appear as dark spots 902. Theimage 905 extracted from the video clip is recorded of 1.2 micron sizepolystyrene beads from Bangs Laboratories, Inc™. Original image 905 wasrecorded with transmitted light from white LED flash light illuminationusing the setup described in FIG. 5.

The band-pass filtered image and the identified bead positions in asingle frame are shown in FIGS. 9B-1 and 9B-2. The beads appear asbright spots 903 after pre-processing with band-pass filtering in theband-pass filtered gray-scale image 903 of FIG. 9B-1. FIG. 9B-2illustrates a zoomed in image 915 of the detected bead positions shownas circles 904. The displacement of the positions of the beads betweentwo consecutive frames was obtained by similar analysis utilized abovein FIGS. 7 and 8. However, in this case where the beads are free to movein the solution, beads are displaced (i.e., travel) by as large as 15pixels in FIGS. 9C-1, 9C-2, and 9C-3. Moreover, the distribution oftheir displacement can be fit nicely to Gaussian distributions in FIGS.9C-1, 9C-2, and 9C-3 for both x and y positions. This (i.e., thedistribution of the x and y displacements for beads free to move in thesolution) is in sharp contrast with the results obtained for staticbeads described earlier in FIGS. 7 and 8. This behavior (thedistribution of the x and y displacements for beads free to move in thesolution) in FIGS. 9C-1, 9C-2, and 9C-3 is expected from the Brownianmotion of the beads. These results in FIGS. 9C-1, 9C-2, and 9C-3 showthat the experimental setup with the ball lens has sufficiently largenumerical aperture and high resolution to resolve individual beads ofsize 1.2 microns.

FIG. 9C-1 is a graph 920 illustrating that the bead x-position shiftedbetween two frames fits nicely to a Gaussian distribution expected forthe random displacement of the beads with the maximum alloweddisplacement of 5 pixels. FIG. 9C-2 is a graph 925 of the distributionwhen the maximum allowed x-shift in pixel position is 15 pixels. FIG.9C-2 also fits nicely to a Gaussian demonstrating that the beads undergorandom displacements expected from their Brownian motion, which can beimaged with the cellular-phone microscope setup described in FIG. 4.FIG. 9C-1 is a graph 930 illustrating that similar behavior occurs forthe y-position shifts in the bead positions.

FIGS. 10A and 10B together illustrate a process 1000 of opticaldetection to determine the absence (i.e., when there is no tethering ofthe micron size polymer beads 30 to the sample surface) or presence(i.e., when there is tethering of micron size polymer beads 30 to thesample surface 10) of a nucleic acid sequence in a sample being testedaccording to an embodiment.

At block 1002, the glass surface or polymer surface of sample cell 10 iscoated with antibody molecules 202. After most of the antibody molecules202 bind to the surface, the sample cell surface 10 may optionally beflushed with buffer solution to remove unbound antibody molecules 202from the solution in sample cell 10.

At block 1005, the sample cell surface 10 is coated with short probe DNAoligomers 205 in which the short probe DNA oligomers 205 are attachedwith anti-digoxigenin molecules 203 on one end which will bind to theantibody molecules 202 coated on the sample cell interior surface 10.

At block 1010, the unknown DNA molecule 210 being tested is biotinylatedon one end (with the second molecule 220) so that the biotinylated endof the DNA molecule 210 can then be attached to the micron size polymerbeads 30 coated with the first molecule 215 (e.g., streptavidin proteinmolecules).

At block 1015, a small amount (e.g., 10-20 microliters (μL) in oneimplementation) of DNA-attached beads 30 is added to the sample cellsurface 10 coated with the short probe DNA oligomers 205.

At block 1020, the Brownian motion of the DNA-attached beads 30 is videomonitored with, e.g., a CCD camera, other detector, and/or the cellularphone 401 in the camera setup in FIGS. 4 and 5.

At block 1025, the cellular phone 401 and/or a computer system 1200 isconfigured to detect the bead positions and follow their track overtime.

At block 1030, the cellular phone 401 and/or a computer system 1200 isconfigured to analyze the tracked bead positions (over a predeterminednumber of consecutive video frames) for restricted Brownian motion bycomparing the tracked bead positions to free Brownian motion behavior(for the same type of beads 30 in the liquid 20 without the unknown DNAmolecule 210 attached and/or for the same type of beads 30 in the liquid20 without the short probe DNA oligomers 205 coated on the samplesurface.

At block 1035, when the cellular phone 401 and/or a computer system 1200recognizes that there is restricted Brownian motion for the trackedbeads 30, the cellular phone 401 and/or a computer system 1200determines that a specific known nucleic acid sequence is present in theunknown DNA molecule 210 being tested which directly corresponds to DNAtethering of the tracked beads 30.

At block 1040, when the cellular phone 401 and/or a computer system 1200recognizes that there is no restricted Brownian motion for the trackedbeads 30, the cellular phone 401 and/or a computer system 1200determines the absence of a specific known nucleic acid sequence fromthe unknown DNA molecule 210 being tested because of no DNA tethering ofthe tracked beads 30.

In one implementation, in performing block 1030, the cellular phone 401and/or a computer system 1200 is configured to calculate the diffusioncoefficient of the bead motion for the tracked beads 30, and thencompare the calculated diffusion coefficient (of the tracked beads 30having the DNA molecule 210 attached) to that of the known diffusioncoefficient (i.e., baseline) for free particle motion (ofparticles/beads having the same or similar size as the tracked beads 30)to determine if there is a statistically significant restricted Brownianfor the tracked beads 30.

When there is no statistically significant restricted Brownian motionfor the tracked beads 30 (as compared to the known diffusion coefficientfor free particle motion), then the cellular phone 401 and/or a computersystem 1200 is configured to determine that there is the absence of aspecific nucleic acid sequence from the unknown DNA molecule 210 beingtested because of no DNA tethering of the tracked beads 30.

However, when there is statistically significant restricted Brownianmotion for the tracked beads 30 (as compared to the known diffusioncoefficient for free particle motion), then the cellular phone 401and/or a computer system 1200 is configured to determine that there isthe specific nucleic acid sequence present in the unknown DNA molecule210 being tested which directly corresponds to DNA tethering of thetracked beads 30.

Now turning to FIGS. 11A and 11B, a method 1100 is provided to determinea presence of a known nucleic acid sequence of a known molecule in asample in which the sample contains a targeted nucleic acid sequence 210according to an embodiment. The known molecule is known in advance.

At block 1102, the glass surface or polymer surface of sample cell 10 iscoated with antibody molecules 202.

At block 1103, the sample cell surface 10 is coated with first shortprobe DNA oligomeric molecules 205 attached with anti-digoxigeninmolecules 203 on one end which will bind to the antibody molecules 202coated on sample cell interior surface 10.

At block 1104, a plurality of beads are coated with second short probeDNA oligomeric molecules 1405.

At block 1105, a plurality of beads 30 coated with second short probeDNA molecules 1405 are mixed with the targeted nucleic acid sequencemolecule 210 (e.g., the DNA/RNA molecule of virus, bacteria, pathogen,plant, human, animal, etc.), and the sample surface 10 is configured tohold a liquid medium 20.

At block 1110, the sample is placed on the sample surface 10, and thesample comprises the liquid medium 20, the plurality of beads 30, andthe targeted nucleic acid sequence molecule 210 being tested for havingthe known nucleic acid sequence of the known molecule, where thetargeted nucleic acid sequence is unknown (at this point).

At block 1115, this mixture is left for sufficient time (e.g., which maybe 30, 30, 90 minutes in one case) enable binding of the targetednucleic acid sequence 210 to the first and second oligomeric DNAmolecules 205 and 1405 attached to the sample surface 10 and pluralityof beads 30 respectively and therefore forming tethers for the beads 30.

At block 1125, the cellular phone 401 (operating as an optical device,e.g., via a video/camera recorder) is configured to video monitor (i.e.,video record) the Brownian motion of the plurality of beads 30.

At block 1130, the cellular phone 401 is configured to determine whetherthe Brownian motion of the plurality of beads 30 in the liquid medium 20is restricted or not restricted over time.

At block 1135, in response to determining (e.g., via the cellular phone401) that the Brownian motion of the plurality of beads 30 isrestricted, the cellular phone 401 is configured to recognize thepresence of the known nucleic acid sequence in the sample, thusindicating that the targeted nucleic acid sequence 210 is the knownnucleic acid sequence.

At block 1140, in response to determining that the Brownian motion isnot restricted, the cellular phone 401 is configured to recognize thatthe known nucleic acid sequence is not present in the sample, thusindicating that the targeted nucleic acid sequence 210 is not the knownnucleic acid sequence.

Determining whether the Brownian motion of the plurality of beads 30 isrestricted or not restricted over time (such as over consecutive videoframes of the recorded video) includes: 1) detecting bead positions ofthe plurality of beads 30 in the liquid medium 20; 2) tracking the beadpositions of the plurality of beads 30 over time to obtain bead motionfor the plurality of beads 30; and 3) comparing the bead motion trackedfor the plurality of beads to free Brownian motion.

Comparing the bead motion tracked for the plurality of beads to freeBrownian motion includes determining that the sample contains the knownnucleic acid sequence based on the bead motion of the plurality of beadsbeing less than the free Brownian motion.

The bead motion of the plurality of beads being less than the freeBrownian motion denotes that the targeted nucleic acid sequence in thesample has tethered the plurality of beads 30 to the sample surface 10.Tethering the plurality of beads 30 to the sample surface restricts thebead motion of the plurality of beads 30. Tethering the plurality ofbeads 30 to the sample surface 10 restricts the bead motion of theplurality of beads to a circular motion.

The targeted nucleic acid sequence in the sample has a first end and asecond end. Tethering the plurality of beads to the sample surface 10comprises the select segment 205 coated on the sample surface 10attaching to the first end of the targeted nucleic acid sequence 210 andcomprises the first molecule 215 coated on the plurality of beads 30attaching to the second end of the targeted nucleic acid sequence.

Comparing the bead motion tracked for the plurality of beads 30 to freeBrownian motion includes determining that the sample does not containthe known nucleic acid sequence based on the bead motion of theplurality of beads 30 corresponding to the free Brownian motion.

Comparing (by the cellular phone 401) the bead motion tracked for theplurality of beads to free Brownian motion includes: 1) determining asample diffusion coefficient of the bead motion tracked for theplurality of beads when the sample of the targeted nucleic acid sequenceis added to liquid medium; 2) determining a baseline diffusioncoefficient for the plurality of beads when no targeted nucleic acidsequence is added to the liquid medium 20; 3) determining that theBrownian motion of the plurality of beads is restricted when the samplediffusion coefficient is less than the baseline diffusion coefficient;4) determining that the Brownian motion of the plurality of beads is notrestricted when the sample diffusion coefficient corresponds to thebaseline diffusion coefficient.

In one implementation, the plurality of beads are in the size range of0.2 to 3.0 microns. The optical device is an electronic communicationdevice having a lens assembly adapter.

Embodiments discussed herein can be utilized in various applications. Assuggested herein, there is a growing need for early and fast diagnosisof flu-like symptoms with point-of-care diagnostic kits either at one'shome or away in a doctor's office such as at an epidemic center. This isalso highlighted by the recent epidemic associated with Ebola virus inAfrica. At one time, it was estimated that the currently infected peoplethat need treatment are between 10,000 and 15,000 in the countriesGuinea, Sierra Leone, and Liberia. It was predicted that this numberwould double every month which will result in nearly 50,000 infectedpeople in one month and 100,000 people in two months (“Containing Ebola:What it would take”, Washington Post, Oct. 9, 2014;(http://apps.washingtonpost.com/g/page/national/containing-ebola-what-it-would-take/1366/).Embodiments based on the restricted Brownian motion of beads can beapplied to develop a cell-phone based diagnostic kit for detecting fluvirus, for example. The viral RNA/DNA can be extracted from the blood orsaliva of the patient after doing the recommended preparation usingcommercially available RNA/DNA extraction kits. As discussed inembodiments, the extracted RNA/DNA is to detect the presence of specificRNA or DNA by the tethering the DNA/RNA forms with the speciallyprepared beads. It is estimated that the diagnosis can probably be donein one to two hours, and the system 480, 580 is completely portable andusable with the cellular phone.

FIG. 12 illustrates an example of a computer system 1200 programed toexecute the features of embodiments discussed. In one case, the computersystem 1200 may be implemented in the camera/video recorder cellularphone 401 discussed herein. Although a camera cellular phone 401 hasbeen discussed, it is understood that embodiments are not limited to ause by a cellular phone. Since portability is a beneficial feature, thecellular phone 401 was discussed. It can be appreciated that other typesof electronic communication devices having camera and/or video recordingcapabilities may be utilized such as a tablet, laptop, video camera,etc. The special lens adapter 400 can be utilized with these electronicdevices if needed.

Various methods, procedures, modules, flow diagrams, tools,applications, circuits, elements, and techniques discussed herein mayalso incorporate and/or utilize the capabilities of the computer 1200.Moreover, capabilities of the computer 1200 may be utilized to implementfeatures of exemplary embodiments discussed herein. One or more of thecapabilities of the computer 1200 may be utilized to implement,incorporate, to connect to, and/or to support any element discussedherein (as understood by one skilled in the art).

Generally, in terms of hardware architecture, the computer 1200 mayinclude one or more processors 1210, computer readable storage memory1220, and one or more input and/or output (I/O) devices 1270 that arecommunicatively coupled via a local interface (not shown). The localinterface can be, for example but not limited to, one or more buses orother wired or wireless connections, as is known in the art. The localinterface may have additional elements, such as controllers, buffers(caches), drivers, repeaters, and receivers, to enable communications.Further, the local interface may include address, control, and/or dataconnections to enable appropriate communications among theaforementioned components.

The processor 1210 is a hardware device for executing software that canbe stored in the memory 1220. The processor 1210 can be virtually anycustom made or commercially available processor, a central processingunit (CPU), a data signal processor (DSP), or an auxiliary processoramong several processors associated with the computer 1200, and theprocessor 1210 may be a semiconductor based microprocessor (in the formof a microchip) or a microprocessor. Note that the memory 1220 can havea distributed architecture, where various components are situated remotefrom one another, but can be accessed by the processor 1210.

The software in the computer readable memory 1220 may include one ormore separate programs, each of which comprises an ordered listing ofexecutable instructions for implementing logical functions. The softwarein the memory 1220 includes a suitable operating system (O/S) 1250,source code 1230, and one or more applications 1260 of the exemplaryembodiments. As illustrated, the application 1260 comprises numerousfunctional components for implementing the features, processes, methods,functions, and operations of the exemplary embodiments. The application1260 of the computer 1200 may represent numerous applications, agents,software components, modules, interfaces, controllers, etc., asdiscussed herein but the application 1260 is not meant to be alimitation.

The operating system 1250 may control the execution of other computerprograms, and provides scheduling, input-output control, file and datamanagement, memory management, and communication control and relatedservices.

The application 1260 may be a source program, executable program (objectcode), script, or any other entity comprising a set of instructions tobe performed. When a source program, then the program is usuallytranslated via a compiler, assembler, interpreter, or the like, whichmay or may not be included within the memory 1220, so as to operateproperly in connection with the O/S 1250. Furthermore, the application1260 can be written as (a) an object oriented programming language,which has classes of data and methods, or (b) a procedure programminglanguage, which has routines, subroutines, and/or functions.

The I/O devices 1270 may include input devices (or peripherals) such as,for example but not limited to, a mouse, keyboard, scanner, microphone,camera, etc. Furthermore, the I/O devices 1270 may also include outputdevices (or peripherals), for example but not limited to, a printer,display, etc. Finally, the I/O devices 1270 may further include devicesthat communicate both inputs and outputs, for instance but not limitedto, a NIC or modulator/demodulator (for accessing remote devices, otherfiles, devices, systems, or a network), a radio frequency (RF) or othertransceiver, a telephonic interface, a bridge, a router, etc. The I/Odevices 1270 also include components for communicating over variousnetworks, such as the Internet or an intranet. The I/O devices 1270 maybe connected to and/or communicate with the processor 1210 utilizingBluetooth connections and cables (via, e.g., Universal Serial Bus (USB)ports, serial ports, parallel ports, FireWire, HDMI (High-DefinitionMultimedia Interface), PCIe, InfiniBand®, or proprietary interfaces,etc.).

The computer 1200 includes one or more antennas 1275 for transmittingand receiving over-the-air-signals (e.g., radio signals such asmicrowave signals, GPS signals, etc.) via a transceiver 1280.

When the computer 1200 is in operation, the processor 1210 is configuredto execute software stored within the memory 1220, to communicate datato and from the memory 1220, and to generally control operations of thecomputer 1200 pursuant to the software. The application 1260 and the O/S1250 are read, in whole or in part, by the processor 1210, perhapsbuffered within the processor 1210, and then executed.

When the application 1260 is implemented in software it should be notedthat the application 1260 can be stored on virtually any computerreadable storage medium for use by or in connection with any computerrelated system or method.

The application 1260 can be embodied in any computer-readable medium foruse by or in connection with an instruction execution system, apparatus,server, or device, such as a computer-based system, processor-containingsystem, or other system that can fetch the instructions from theinstruction execution system, apparatus, or device and execute theinstructions.

In exemplary embodiments, where the application 1260 is implemented inhardware, the application 1260 can be implemented with any one or acombination of the following technologies, which are each well known inthe art: a discrete logic circuit(s) having logic gates for implementinglogic functions upon data signals, an application specific integratedcircuit (ASIC) having appropriate combinational logic gates, aprogrammable gate array(s) (PGA), a field programmable gate array(FPGA), etc.

It is understood that the computer 1200 includes non-limiting examplesof software and hardware components that may be included in variousdevices, servers, and systems discussed herein, and it is understoodthat additional software and hardware components may be included in thevarious devices and systems discussed in exemplary embodiments.

As will be appreciated by one skilled in the art, one or more aspects ofthe present invention may be embodied as a system, method or computerprogram product. Accordingly, one or more aspects of the presentinvention may take the form of an entirely hardware embodiment, anentirely software embodiment (including firmware, resident software,micro-code, etc.) or an embodiment combining software and hardwareaspects that may all generally be referred to herein as a “circuit,”“module” or “system”. Furthermore, one or more aspects of the presentinvention may take the form of a computer program product embodied inone or more computer readable medium(s) having computer readable programcode embodied thereon.

Any combination of one or more computer readable medium(s) may beutilized. The computer readable medium may be a computer readablestorage medium. A computer readable storage medium may be, for example,but not limited to, an electronic, magnetic, optical, electromagnetic,infrared or semiconductor system, apparatus, or device, or any suitablecombination of the foregoing. More specific examples (a non-exhaustivelist) of the computer readable storage medium include the following: anelectrical connection having one or more wires, a portable computerdiskette, a hard disk, a random access memory (RAM), a read-only memory(ROM), an erasable programmable read-only memory (EPROM or Flashmemory), an optical fiber, a portable compact disc read-only memory(CD-ROM), an optical storage device, a magnetic storage device, or anysuitable combination of the foregoing. In the context of this document,a computer readable storage medium may be any tangible medium that cancontain or store a program for use by or in connection with aninstruction execution system, apparatus, or device.

The descriptions of the various embodiments of the present inventionhave been presented for purposes of illustration, but are not intendedto be exhaustive or limited to the embodiments disclosed. Manymodifications and variations will be apparent to those of ordinary skillin the art without departing from the scope and spirit of the describedembodiments. The terminology used herein was chosen to best explain theprinciples of the embodiments, the practical application or technicalimprovement over technologies found in the marketplace, or to enableothers of ordinary skill in the art to understand the embodimentsdisclosed herein.

The present invention may be a system, a method, and/or a computerprogram product. The computer program product may include a computerreadable storage medium (or media) having computer readable programinstructions thereon for causing a processor to carry out aspects of thepresent invention.

The computer readable storage medium can be a tangible device that canretain and store instructions for use by an instruction executiondevice. The computer readable storage medium may be, for example, but isnot limited to, an electronic storage device, a magnetic storage device,an optical storage device, an electromagnetic storage device, asemiconductor storage device, or any suitable combination of theforegoing. A non-exhaustive list of more specific examples of thecomputer readable storage medium includes the following: a portablecomputer diskette, a hard disk, a random access memory (RAM), aread-only memory (ROM), an erasable programmable read-only memory (EPROMor Flash memory), a static random access memory (SRAM), a portablecompact disc read-only memory (CD-ROM), a digital versatile disk (DVD),a memory stick, a floppy disk, a mechanically encoded device such aspunch-cards or raised structures in a groove having instructionsrecorded thereon, and any suitable combination of the foregoing. Acomputer readable storage medium, as used herein, is not to be construedas being transitory signals per se, such as radio waves or other freelypropagating electromagnetic waves, electromagnetic waves propagatingthrough a waveguide or other transmission media (e.g., light pulsespassing through a fiber-optic cable), or electrical signals transmittedthrough a wire.

Computer readable program instructions described herein can bedownloaded to respective computing/processing devices from a computerreadable storage medium or to an external computer or external storagedevice via a network, for example, the Internet, a local area network, awide area network and/or a wireless network. The network may comprisecopper transmission cables, optical transmission fibers, wirelesstransmission, routers, firewalls, switches, gateway computers and/oredge servers. A network adapter card or network interface in eachcomputing/processing device receives computer readable programinstructions from the network and forwards the computer readable programinstructions for storage in a computer readable storage medium withinthe respective computing/processing device.

Computer readable program instructions for carrying out operations ofthe present invention may be assembler instructions,instruction-set-architecture (ISA) instructions, machine instructions,machine dependent instructions, microcode, firmware instructions,state-setting data, or either source code or object code written in anycombination of one or more programming languages, including an objectoriented programming language such as Smalltalk, C++ or the like, andconventional procedural programming languages, such as the “C”programming language or similar programming languages. The computerreadable program instructions may execute entirely on the user'scomputer, partly on the user's computer, as a stand-alone softwarepackage, and partly on a remote computer or entirely on the remotecomputer or server. In the latter scenario, the remote computer may beconnected to the user's computer through any type of network, includinga local area network (LAN) or a wide area network (WAN), or theconnection may be made to an external computer (for example, through theInternet using an Internet Service Provider). In some embodiments,electronic circuitry including, for example, programmable logiccircuitry, field-programmable gate arrays (FPGA), or programmable logicarrays (PLA) may execute the computer readable program instructions byutilizing state information of the computer readable programinstructions to personalize the electronic circuitry, in order toperform aspects of the present invention.

Aspects of the present invention are described herein with reference toflowchart illustrations and/or block diagrams of methods, apparatus(systems), and computer program products according to embodiments of theinvention. It will be understood that each block of the flowchartillustrations and/or block diagrams, and combinations of blocks in theflowchart illustrations and/or block diagrams, can be implemented bycomputer readable program instructions.

These computer readable program instructions may be provided to aprocessor of a general purpose computer, special purpose computer, orother programmable data processing apparatus to produce a machine, suchthat the instructions, which execute via the processor of the computeror other programmable data processing apparatus, create means forimplementing the functions/acts specified in the flowchart and/or blockdiagram block or blocks. These computer readable program instructionsmay also be stored in a computer readable storage medium that can directa computer, a programmable data processing apparatus, and/or otherdevices to function in a particular manner, such that the computerreadable storage medium having instructions stored therein comprises anarticle of manufacture including instructions which implement aspects ofthe function/act specified in the flowchart and/or block diagram blockor blocks.

The computer readable program instructions may also be loaded onto acomputer, other programmable data processing apparatus, or other deviceto cause a series of operational steps to be performed on the computer,other programmable apparatus or other device to produce a computerimplemented process, such that the instructions which execute on thecomputer, other programmable apparatus, or other device implement thefunctions/acts specified in the flowchart and/or block diagram block orblocks.

The flowchart and block diagrams in the Figures illustrate thearchitecture, functionality, and operation of possible implementationsof systems, methods, and computer program products according to variousembodiments of the present invention. In this regard, each block in theflowchart or block diagrams may represent a module, segment, or portionof instructions, which comprises one or more executable instructions forimplementing the specified logical function(s). In some alternativeimplementations, the functions noted in the block may occur out of theorder noted in the figures. For example, two blocks shown in successionmay, in fact, be executed substantially concurrently, or the blocks maysometimes be executed in the reverse order, depending upon thefunctionality involved. It will also be noted that each block of theblock diagrams and/or flowchart illustration, and combinations of blocksin the block diagrams and/or flowchart illustration, can be implementedby special purpose hardware-based systems that perform the specifiedfunctions or acts or carry out combinations of special purpose hardwareand computer instructions.

What is claimed is:
 1. A method to determine a presence of a knownnucleic acid sequence of a known molecule in a sample in which thesample contains a targeted nucleic acid sequence, the method comprising:coating a sample surface with a select segment of the known molecule,wherein the sample surface is configured to hold a liquid medium;coating a plurality of beads with a first molecule; attaching thetargeted nucleic acid sequence to a second molecule that is configuredto bind to the first molecule, such that the targeted nucleic acidsequence is attached to the plurality of beads via the first and secondmolecules; placing the sample on the sample surface, the sampleincluding the liquid medium, the plurality of beads, and the targetednucleic acid sequence being tested for having the known nucleic acidsequence of the known molecule, the targeted nucleic acid sequence beingunknown; video monitoring a Brownian motion of the plurality of beadswith an optical device; determining whether the Brownian motion of theplurality of beads in the liquid medium is restricted or not restrictedover time; in response to determining that the Brownian motion of theplurality of beads is restricted, recognizing the presence of the knownnucleic acid sequence in the sample, thus indicating that the targetednucleic acid sequence is the known nucleic acid sequence; and inresponse to determining that the Brownian motion is not restricted,recognizing that the known nucleic acid sequence is not present in thesample, thus indicating that the targeted nucleic acid sequence is notthe known nucleic acid sequence.
 2. The method of claim 1, whereindetermining whether the Brownian motion of the plurality of beads isrestricted or not restricted over time includes: detecting beadpositions of the plurality of beads in the liquid medium; tracking thebead positions of the plurality of beads over time to obtain bead motionfor the plurality of beads; and comparing the bead motion tracked forthe plurality of beads to a free Brownian motion.
 3. The method of claim2, wherein comparing the bead motion tracked for the plurality of beadsto the free Brownian motion includes determining that the samplecontains the known nucleic acid sequence based on the bead motion of theplurality of beads being less than the free Brownian motion.
 4. Themethod of claim 3, wherein the bead motion of the plurality of beadsbeing less than the free Brownian motion denotes that the targetednucleic acid sequence in the sample has tethered the plurality of beadsto the sample surface.
 5. The method of claim 4, wherein tethering theplurality of beads to the sample surface restricts the bead motion ofthe plurality of beads.
 6. The method of claim 4, wherein tethering theplurality of beads to the sample surface restricts the bead motion ofthe plurality of beads to a circular motion.
 7. The method of claim 4,wherein the targeted nucleic acid sequence in the sample has a first endand a second end; wherein tethering the plurality of beads to the samplesurface comprises the select segment coated on the sample surfaceattaching to the first end of the targeted nucleic acid sequence andcomprises the first molecule coated on the plurality of beads attachingto the second end of the targeted nucleic acid sequence.
 8. The methodof claim 2, wherein comparing the bead motion tracked for the pluralityof beads to the free Brownian motion includes determining that thesample does not contain the known nucleic acid sequence based on thebead motion of the plurality of beads corresponding to the free Brownianmotion.
 9. The method of claim 2, wherein comparing the bead motiontracked for the plurality of beads to the free Brownian motion includes:determining a sample diffusion coefficient of the bead motion trackedfor the plurality of beads when the sample of the targeted nucleic acidsequence is added to the liquid medium; determining a baseline diffusioncoefficient for the plurality of beads when no targeted nucleic acidsequence is added to the liquid medium; determining that the Brownianmotion of the plurality of beads is restricted when the sample diffusioncoefficient is less than the baseline diffusion coefficient; determiningthat the Brownian motion of the plurality of beads is not restrictedwhen the sample diffusion coefficient corresponds to the baselinediffusion coefficient.
 10. The method of claim 1, wherein the pluralityof beads are micron size; and wherein the optical device is anelectronic communication device having a lens assembly adapter.